The food industry has traditionally used sugar (sucrose) as a sweetening agent. There are many synthetic and natural sources of sweeteners available in the market. The most common high intensity synthetic sweeteners in the world market are acesulfame-K, aspartame, neotame, saccharin and sucralose. The natural sweeteners available commercially include thaumatin, glycyrrhizin, xylitol, mogroside etc.
The increased consumption of high calorie sugars has resulted in several nutritional and medical problems, such as obesity, diabetes and cardiovascular diseases. Anton, S D et al., Appetite 55(1): 37-43 (2010). Dietary and health demands are continuing to expand the market for sweeteners as alternatives to sucrose. Therefore, there is a continuing search for non-nutritive, low-calorific, non-toxic and high intensity sweeteners, which can be used as sucrose alternatives.
Plants are gaining importance as major sources of high potency sweeteners in the growing natural food market. The plant Stevia rebaudiana Bertoni (family Asteraceae) commonly known as “a sweet herb” has been widely cultivated and is known as “a sweetener of the future.” (a) Wood, H B et al., J. Org. Chem. 20(7):875-883 (1955); (b) Tanaka, O, Trends Anal. Chem. 1:246-248 (1982); (c) Geuns, JMC Phytochemistry 64(5):913-921 (2003); (d) Wallin, H, 63rd JECFA (FAO) 1-5 (2004); (e) Esmat Abou-Arab, A et al., Afr. J. Food Sci. 4(5):269-281 (2010); (f) Madan, S et al., Indian J. Nat. Prod. Resor. 1(3):267-286 (2010). The leaves of Stevia rebaudiana accumulate sweet-tasting steviosides, as the main diterpene glycosides with the common backbone of steviol. These terpenoids differ in the number of carbohydrate residues at C13 and C19 positions of the steviol skeleton. These steviol glycosides are 250-300 times sweeter than cane sugar with excellent compatibility with tea, coffee and other hot as well as cold beverages. (a) Clos, J F et al., J. Agric. Food Chem. 56:8507-8513 (2008). These sweet diterpene glycosides are non-caloric, non-nutritive, non-toxic and non-mutagenic in nature.
Among the nine steviol glycosides, Stevioside and Rebaudioside-A are the major constituents of the native plant S. rebaudiana. Kovylyaeva et al. Chem. Nat. Compd. 43(1):81-85 (2007). When eaten regularly they decrease the content of blood sugar and cholesterol in the body, stimulate cell regeneration and strengthen blood vessels, thus these sweeteners are industrially/commercially important. (a) Chatsudthipong, V et al., Pharmacol Ther. 121(1):41-54 (2009); (b) Geeraert, B et al., Int J Obes. 34(3):569-577 (2010). The steviosides are the most exhaustively utilized diterpene glycosides that are used as sweeteners in both the food and the beverage industries. Apart from sweetening, steviosides also exert cardio-tonic and anti-oxidant effects, which is an absolute necessity in endurance sports. In addition, steviosides also exhibit promising therapeutic value as choleretic, anti-inflammatory, anticancer, diuretic agents and also prevent ulcers in the gastrointestinal tract. (a) Kennelly, E J. Sweet and non-sweet constituents of Stevia rebaudiana (Bertoni). In: Stevia, the genus Stevia. Medicinal and Aromatic plants -Industrial profiles. Kinghom A D (ed), Taylor and Francis, London and NY. 1:68-85 (2002); (b) Gregersen, S. Metabolism 53(1):73-76 (2004); (c) Wolwer-Rieck, U et al., J. Agric. Food Chem. 58:12216-12220 (2010); (d) Stevioside: Technology, Applications and Health. De, S; Mondal, S; Banerjee, S. Wiley and Sons, Copyright.(2013).
Despite wide therapeutic application of steviosides, their uses are restricted for human consumption due to their bitter after-taste and astringency. The grassy taste associated with the presence of some alkaloid/diterpene impurities, limits their application in the food and the pharmaceutical industries.
The sweetening potency and edulcorant properties of the different steviol glycosides present in the leaves of the Stevia plant differ from one another, thus the quality of the commercial product depends on the proportion of the steviol glycosides in the mixture. Among the important factors in producing a pleasant taste are the percentage/ratio of Stevioside and Rebaudioside-A present in the total extract of steviol glycosides. Attempts are made to improve the sweetness of steviosides by adding at least one natural sweetener like sucrose, glucose and fructose to them by glycosidation or by membrane separation of impurities to obtain high purity steviosides. (a) Kirk, D E et al., J Food Sci. 48:1663-1666 (1983); (b) Fuh, W S et al., J. Food Sci. 55(5):1454-1457 (1990); (c) Zhang, S Q et al. Food Research International. 33(7):617-620 (2000); (d) Rajasekaran, T et al., Food Biotech. 22(2):179-188 (2002); (e) Rajasekaran, T et al., J. Sci. Food Agric. 87:420-424 (2007); (f) Esmat Abou-Arab, A et al., Afr. J. Food Sci. 4(5): 269-281 (2010).
There has been a remarkable resurgence of interest in natural product research over the last decade or so, starting from extraction to determination of the structures of purified natural products, and their biological activity. (a) Sarker, S D; Latif, Z; Gray, A I. Methods in Biotechnology: Natural Product Isolation, Humana Press, New Jersey, USA, (2006); (b) Chemat, F; Strube, J. Green Extraction of Natural Products: Theory and Practice. Wiley-VCH, Verlag GmbH & Co.KGaA (2015). Prior to any isolation and purification work, natural products have to be extracted/released from the biomass. The choice of extraction procedure depends on the nature of the source material and the compounds to be isolated. The design of green and sustainable extraction methods of natural products is currently a hot research topic in the multidisciplinary area of Applied Chemistry and Food Technology. Bioactive compounds in plants are typically present at low concentrations. ‘Classic’ solvent-based extraction of phytochemicals procedures (e.g. maceration, percolation, Soxhlet extraction, extraction under reflux, steam distillation and super critical carbon dioxide extraction) are still applied widely in photochemistry despite the fact that these methodologies lack reproducibility and are both time and solvent consuming. (a) Liu, J et al., J. Chromatogr. Sci. 35(9):446-450 (1997); (b) Jaitak, V et al., Phytochem. Anal. 20(3):240-245 (2009); (c) Erkucuk, A. et al., J. Supercrit. Fluids. 51(1):29-35 (2009); (d) Mustafa, A. et al., Anal. Chim. Acta. 703(1):8-18 (2011); (e) Jentzer, J B et al., Food Chem. 166:561-567 (2015). Solvent based extraction of bioactives often suffers from low extraction yields, requires long extraction times and decreased product quality.
Recently, enzyme-assisted extraction methods have been reported for the extraction of plant-based bioactives. (a) Ruiz-Terán, F et al., J. Agric. Food Chem. 49(11):5207-5209 (2001); (b) Sowbhagya, H B et al.; Crit. Rev. Food. Sci. Nutr. 50:146-161 (2010); (c) Puri, M. et al., Food Chem. 132:1113-1120 (2012). Enzyme-based extraction of bioactive compounds from plants is a potential alternative to well known conventional solvent based extraction methodologies. This improved green and eco-friendly extraction process increases the effective solvent pre-treatment, reduces the amount of solvent needed for extraction and increases the yield of extractable compounds. (a) Chemat, F et al., Int J. Mol Sci. 13(7):8615-8627 (2012); (b) Li, J et al., Bioresour. Technol. 155:258-265 (2014). Food-grade hydrolases such as lipases, proteases, cellulases, hemicellulases, pectinases and thermolysin are widely used in processing and clarification, and improve the extractability of the desired compounds.
In recent years there has been an increasing demand for natural, non-nutritive, high intensity sweeteners with low-calorie value, as a sugar substitute. Extracts of the leaves of Stevia rebaudiana have been known for their sweet taste. Stevioside and Rebaudioside-A are the two major diterpenoid glucosides components present in the leaf extracts of Stevia. These glycosides are 300 times sweeter than sugar and also exhibit wide therapeutic activity. The potential widespread use of the steviosides requires an easy and effective extraction methodology. There has been abundant published/patented literature on the extraction and isolation of steviosides from dried stevia leaves. The conventional methods of isolation and purification processes of steviosides involves aqueous, supercritical fluid, microwave and metallic ion extraction methodologies, and the purification processes of steviosides involves long extraction and purification procedures by using different chromatographic techniques like resins and zeolites, membrane separation and electrophoresis methodologies. All these available procedures of extraction processes are having the draw backs like low yields, use of expensive resins and metal ions, some of these processes release noxious residues that are harmful for health and are not eco-friendly. Thus the process developed in isolation of steviosides are expensive and are not eco-friendly; therefore a simple procedures for the isolation of good quality, high yields, with improved organoleptic properties of steviosides is a present day challenging problem. (a) Haga, T; Ise, R; Kobayashi, T. Japanese patent 51-131900 (1976); (b) Kohda, H et al., Phytochem. 15:981-983 (1976); (c) Ishizone, H. Japan Kokai Pat 79,12,400 (1979); (d) Dobberstein, R H; Ahmed, M S. U.S. Pat. No. 4,361,697 (1982); (e) Alvarez, M; Couto, ACC. Brasil Pat. 84,02,752 (1984); (f) Kumar, S. U.S. Pat. No. 4,599,403 (1986); (g) Giovanetto, R H. U.S. Pat. No. 4,892,938 (1990); (h) Lobov, S V et al., Agric. Biol. Chem. 55:2959-2965 (1991); (i) Tan, S; Ueki, H. Jap. Pat. 06-007108 (1994); (j) Moraes, E P et al., Acta Scientiar. 23(6):1375-1380 (2001); (k) Kotesh, K J; Babu, G D K et al. US Patent 2006/0142555A1 (June 2006) 11/022277/US; dated: Dec. 23, 2004; (l) De Oliviera, B H et al. J. Biotech. 131(1):92-96 (2007); (m) Abelyan, V H; Ghochikyan, Y T et al. US Pat. 7838044B2, U.S. Ser. No. 11/016,781 (2010); (n) Puri, Met al., Biotech. Adv. 29(6):781-791 (2011); (o) Wehrli, C. EP 2526195 A1 (2012); (q) Puri, M. et al., Food Chem. 132(3):1113-1120 (2012); (r) G. Roopa Reddy, G, A.Bhaskar Rao, A, and Yadav J S (Indian patent No: 0033NF 2011 dated Sep. 2, 2011.) Indian patent Application no: 2288/del/2011. (s) Rao, A B et al.; Adv Biosci. Biotechnol. 3:327-335 (2012); (t) Das, A et al., Sep. Purif. Technol. 144 (C):8-15 (2015).
Accordingly, there is a need to develop an improved enzyme-assisted extraction process of steviosides from the leaves of Stevia rebaudiana Bertoni and to identify the necessary experimental conditions in isolation of total steviosides.